A major challenge remaining is the reconstruction of damaged and diseased neural pathways. Toward this end, biomaterials have been examined as bridging devices to support directed nerve outgrowth from regenerating or transplanted neurons. A fundamental challenge that remains is in understanding how to engineer bridges that are clinically useful. The proposed project builds on two observations: 1) aligned scar-like astrocytes support and direct neural outgrowth while misaligned astrocytes display minimal outgrowth even though they display similar surface cues, and 2) neurite outgrowth velocity increases when neurons encounter mixtures of permissive ligands and inhibitory proteoglycans presented in a non-physiological pattern. Based on these observations we formulated the central hypothesis of the proposed research: the local composition and spatial distribution of permissive and inhibitory ligands are the signals that underlie directed outgrowth. by astrocytes. Moreover, we believe it is possible to mimic the spatial distribution of permissive and inhibitory molecules on biomaterials, and that such surfaces could be functionally equivalent to astrocytes coated surfaces in terms of supporting and directing neuronal outgrowth. We will test this hypothesis by: (1) mapping the type and distribution of permissive and inhibitory molecules on aligned astrocyte monolayers using high-resolution microscopy to create a large area map that will aid in the design of a new biologically inspired bridging surface. By designing novel biomimetic structures and interfaces presenting permissive and inhibitory molecules with controlled local composition and spatial distribution we will study how such signals on sub-micrometer length-scales influence axonal outgrowth. (2) We propose to study the role(s) that carbohydrates moieties of the three major proteoglycan families play on directing axonal outgrowth using the novel biomimetic structures as test beds. This will be accomplished using two different routes: using (a) enzymatically modified proteoglycans, or (b) glycosaminoglycans re-assembled from oligosaccharide units synthesized from a precursor using recombinant enzymes. Both types of modifications (a) and (b) then will be tested using axonal outgrowth assays. Finally, (3) we will examine the biomimetic structure under conditions likely encountered following implantation in patients, by using a model system that exposes these surfaces to resting and activated macrophages isolated from CNS. PUBLIC HEALTH RELEVANCE: Among the most debilitating and costly human ailments are injuries and diseases of the nervous system. They affect millions of people in the US and represent a large part of the total national health care cost. Currently there are a limited number of available therapies, none of which restore function to injured neurons of the central nervous system. Numerous studies in animals and man strongly suggest that restorative therapies based on cell transplantation are feasible. A major challenge remains is the reconstruction of damaged and diseased neural pathways. Toward this end, biomaterials have been examined as bridging devices to support directed nerve outgrowth from regenerating neurons. A fundamental challenge involves understanding how to engineer such biomaterial bridges. The proposed project's main objective is to evaluate the construction of such bridging devices based on astrocyte-derived distribution of modified neuron-directing molecules from the nervous system.